SYNTHESIS OF THE ISOAMYL ESTER OF 7-N-ACETYL-ll l -HYDROXY-3 1 DEMETHYLLAVENDAMYCIN
An Honors Thesis (HONRS 499)
by
Thomas J. Eads
Thesis Advisor
Dr. Mohammad Behforouz
Ball State university
Muncie, Indiana
April 27, 1992
Date of Graduation: May 2, 1992
-
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"'Ole5 ' 5
L. D ,
;)..J.f'Z-!
.7 l /
i qCrOl,
PURPOSE OF THESIS
.£13
The purpose of this project is to synthesize the isoamyl ester
of
7-N-acetyl-ll'-hydroxy-3'demethyllavendamycin
potentially be an antitumor drug.
which
may
Similar compounds have been
previously made in our laboratory, but this new compound has two
distinctly different groups attached to the N-acetyllavendamycin
backbone.
These groups will hopefully increase the solubility of
the compound in water, thus making it a more practical drug for use
in humans, as well as increasing its selectivity in destroying the
cancer cells relative to the normal cells.
be
sent to Eli Lilly
for testing first
The new compound will
in vitro,
animals to determine its selectivity and toxicity.
and then in
Lavendamycin-
based drugs offer some hope in treating certain kinds of human
cancers for which there are no drugs at the present time.
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ACKNOWLEDGEMENTS
I
would I ike to thank Dr.
Mohammad Behforouz for being an
excellent instructor, as well as a patient supervisor.
He always
made sure that I understood what I was doing!
Wen Cai
gave me more help than I
appreciated her help,
and I
"keeping me on my toes".
ever could ask
for.
I
am especially grateful to her for
This synthesis was carried out much
easier with her perfectionist style.
Thanks .also to Sandy, Mohammad, and Charmaine for helping me
wi th some of those very necessary "day to day" tasks.
Also, thanks
to
spectroscopy
David
analyses.
Bir
for
your help
on
the
nmr and
mass
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TABLE OF CONTENTS
page
HISTORICAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . • . . . . . .
1
La""endamycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Pharmacological Studies ...........•..•..•...... 3
Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Proposal for New Compound ...............•....••....
5
RESULTS AND DISCUSSION ......................••...••...... 6
Synthesis of the Isoamyl Ester of 7-N-Acetyl-11'hydroxy-3'-demethyllavendamycin ...•....•....... 6
Synthesis of 5-Hydroxytryptophan isoamyl ester ...... 7
preparation of 7-Acetamido-2-formylquinoline5,8-diane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . • . . 10
I.
I I .
GENERAL INFORMATION . . . . . . . . . . . . . . . . • . . . . . • . . . . . . 10
PROCEDURE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
11
A. Preparation of 5,7-Dinitro-8-hydroxy-2methylquinoline ............................. 11
B. Preparation of 5,7-Diacetamido-2-methyl8-acetoxyquinoline .............•••....•.•... 13
C. Preparation of 7-Acetamido-2-methylquinoline5 / B-diane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
D. Preparation of 7-Acetamido-2-formylquinoline5,8-dione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
E. Synthesis of 5-Hydroxytryptophan
isoamyl ester................................ 18
F. Synthesis of the Isoamyl Ester of 7-N-Acetyl11'-hydroxy-3'-demethyllavendamycin ...... 20
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REFERENCES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
APPENDIX ........................ '. . . . . .. .. . .. . .. . . .. . .. .. . . .. .. .. .. .. . .. . .. .. ... 23
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HISTORICAL
LAVENDAXYCIN
I. BACKGROUND
The ant.i tumor,
antibiotic Lavendamycin
(1a)
bears a
close
resemblance to the potent antitumor, antibiotic streptonigrin (2).
Lavendamycin
was
first
lavendulae strain c22030.
solid,
rnp
>
300·,
with
isolated
in
1981
from
streptomyces
Lavendamycin was obtained as a dark, red
only
limited
solubility
in
organic
solvents. 1 As an antibiotic, Lavendamycin is generally less potent
than
streponigrin.
The
exceptions
lie
in
the
lavendamycin
inhibition of Trichophton and Microsporum.
0
H3 CO
C0 2H
H2N
0
1
CH 3
1
N
H
o
compound
Rz
B1
14
B3
1a
NH2
H
H
CH3
1b
NH z
CH3
H
CH3
4
NHAc
CH3
H
CH 3
5
NHAc
OH
(CH 2) 2CH (CH3 ) 2
H
Table 12
minimum inhibitory concentration
Organism
Trichophyton rub rum
strain
(~g/ml)
Lavendamycin
streptonigrin
WW
0.5
8
Trichophyton mentagrophytes
A-9870
0.5
4
Microsporum canis
A-9872
0.5
16
Microsporum canis
A-22494
0.25
2
4
The
potential
clinical
usage
of
both
Lavendamycin
and
streptonigrin as antitumor drugs has been unsuccessful due to their
toxicity.2
Lavendamycin has a B-carboline backbone (3) .
B-carbolines and
their derivatives possess a number of behavioral and neurochemical
activities. 3
II.
PHARMACOLOGICAL STUDIES
In
an
in
vitro
drug
screen,
N-acetyllavendamycin
methyl
ester (4) had shown high selective cytotoxicity for ras k transformed
epithelial cells, compared to the normal cells.
The ras k oncogene
is associated with 90% of pancreatic solid tumors in humans, 60% of
colon
tumors,
selective
and
30% of· breast cancer tumors.
cytotoxicity
advancement,
as
there
compound
is unprecedented
and marks
an
is
drug
cures
currently
tumors. 4
-
r
3
no
that
(4)
exciting
these
-
III.
SYNTHESIS
In 1984,
the first total synthesis of Lavendamycin methyl
ester(lb) was reported by a team at the University of Rochester. 5
The precursor used was B-methyl tryptophan.
Recently Behforouz and
co-workers have
for the synthesis of
found an efficient route
lavendamycin methyl ester(lb).6
A.
Synthesis of Lavendamycin methyl esterC1b)
and its N-
Acetyl Derivative(4)
The new and efficient route referred to above was carried out
by
the
Pictet-Spengler
condensation
of
aldehyde (8)
with
B-
methyl tryptophan methyl ester(10) in refluxing xylene to give NAcetyllavendamycin methyl ester (4) in 79% yield.
in
a
mixture
of
trifluoroacetic
acid
and
Hydrolysis of (4)
water
produced
lavendamycin methyl ester(lb) in 91% yield (Scheme 1).3
o
AcHN
o
CHO
+
xylene
o
AcHN
SCHEME I
-
4
o
Ib
IV.
PROPOSAL FOR THE SYNTHESIS OF THE NEW COMPOUND 7-N-ACETYL-ll, HYDROXY-3'-DEMETHYLLAVENDAMYCIN ISOAMYL ESTER(S)
Lavendamycin methyl ester is a very insoluble compound and
consequently,
it is difficult to be introduced into animals for
screening
tests.
More
soluble
analogs
of
this
compound
are
desired.
The addition of a hydroxyl group to the lavendamycin
backbone will hopefully increase the solubility of the compound in
water, making it more suitable for introduction into animals.
The
location of the hydroxyl group also makes the compound similar to
the naturally occurring Serotonin(6) found in the human brain.
isoamyl group at the C-2
selective cytotoxicity.
I
The
position will hopefully increase the
The antitumor activity of compound (5) and
a number of other lavendamycin analogs prepared by other members of
our
research
compared.
be
group
will
be
studied,
and
the
results
be
Based on this study, the minimum potent structure will
determined,
and
possibly
the
mechanism
of
the
lavendamycin against cancer cells will be clarified.
HO
-
will
5
action
of
RESOLTS AND DISCOSSION
SNTHESIS OF THE ISOAMYL ESTER OF 7-N-ACETYL-ll'-HYDROXY-3'DEMETHYLLAVENDAMYCIN
The maj c)r obj ecti ve of this research was the synthesis of the
isoamyl esterof 7-N-acetyl-11 '-hydroxy-3 '-demethyllavend amycin(S).
This compound was produced through the Pictet-Spengler condensation
of
S-Hydroxytryptophan
isoamyl
formylquinoline-S,8-dione(8)
hours.
ester(7)
with
7-Acetamido-2-
in refluxing dry acetonitrile for 19
This procedure gave a 6.9% yield of a red solid material
according to the following scheme (scheme II).
o
HO
+
AcHN
CHO
o
acetonitrile.
o
2
AcHN
SCHEME II
OH
Thin Layer Chromatography (silica gel,
showed the compound to be pure (Rf=0.4).
acetyl-11'-hydroxy-3'-demethyllavendamycin
,---
confirmed by nmr.
CH2Cl 2
-
MeOH, 100:1)
The structure of 7-Nisoamyl
ester
was
The nmr showed traces of unreacted ester, however.
6
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SYNTHESIS OF 5-HYDROXYTRYPTOPHAN ISOAMYL BSTER
5-Hydroxytryptophan isoamyl ester (7) was prepared according to
a similar method described for the synthesis of B-methyltryptophan
methyl ester.7
The following scheme was used (scheme III).
HO
HO
1) HCl isoamyl alcohol
@ 145-150·C
z
SCHEME III
Condensation of
~
with an excess of isoamyl alcohol in the
presence of dry hydrogen chloride at 145-150·C for 24 hours gave
ester 2
in
80% yield as
a
dark brown semisolid.
Thin layer
chromatography (silica gel, EtOAc/MeOH 9:1) showed the compound to
be pure (Rf=0.51).
The structure was confirmed by nmr,
mass spectroscopy.
7
ir, and
-
PREPARATION OF 7-ACETAMIDO-2-FORMYLQUINOLINE-5,8-DIOHB(8)
7-Acetamino-2-formylquDinoline-5,8-dione(8)
according to the following scheme (scheme IV).
was
prepared
The procedure was
similar to that described by A.G. Richardson. 8
..
HN0 3 /H zS0 4
CH 3
Hz. HCI
CH 3
N0 2
OH
Pd/C
•
OH
OH
12 a
12
11
NHAe
AC 20
NaZS03
NaOAc
•
0
KZCrZ 0 7
CH 3
ACHN
HOAc
OAe
0
1,4 dioxane
•
CHO
ACHN
0
J.
,-..
I
•
CH 3
ACHN
0
13
SeOz
CH 3
NH2
(SCHEME IV)
8
14
Compc;mnd 11 was treated with 70% HN03 -HzS04 at 0-5·C for 90
minutes to give a 58% yield of compound 12 as a bright yellow
solid.
Reduction of 12 with molecular hydrogen in the presence of 5%
Palladium on charcoal at 25 ° C for
mixture.
four hours gave a
dark red
The resulting mixture was filtered off and the filtrate
was treated with excess acetic anhydride in the presence of sodium
sulfite and sodium acetate to give an 80% yield of compound 11 as
a white solid (m.p. 255°C).
Oxidation
of
compound
13
in
acetic
acid
by
potassium
dichromate for 20 hours at 25°C gave a 56% yield of compound 14 as
an orange/yellow solid.
Oxidation of compound 14 in 1,4-dioxane by selenium oxide for
24 hours at reflux gave a 73% yield of compound .§. as a yellow
solid.
9
EXPERIIlENTAL:
I. GENERAL INFORMATION
RBAGEN'l'S: 8-Hydroxyquinalidine, palladium on charcoal (5%), 5-
Hydroxy tryptophan
SOLVEN'l'S: Acetonitrile, 1,4-Dioxane, and Isoamyl alcohol were
dried and distilled before use.
Chloroform, ethyl acetate, ethyl
ether, dichloromethane, 95% ethanol, and methanol were used without
distillation.
MELTING POINTS:
Melting points were performed with a Thomas
Hoover capillary melting point apparatus, and are not corrected.
NUCLEAR MAGNETIC RESONANCE SPECTRA ('H NHR):
NMR spectra were
recorded on a Varian Gemini 200 in deuterated chloroform (CDCl 3 )
with
TMS
as
the
internal
standard.
The
chemical
shifts
are
reported in 6 values.
TLC I
THIN LAYER CHROMATOGRAPHY:
S
were performed on Kodak
silica gel sheets containing a fluorescent indicator.
INFRARED SPECTROSCOPY:
IRis were performed on a Nicolet 5ZDX
FT-IR spectrometer.
MASS SPECTROSCOPY:
Mass spectra were recorded on an Extrel
ELQ 400 mass spectrometer.
PLATE CHROMATOGRAPHY:
Plate chromatographies were performed
on Analtech alumina gel thin layer chromatography plates.
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I
10
II. PROCEDURE
A.
PREPARATION OF 5,7- DINITRO-8-HYDROXY-2-METHYLQUINOLINE(12)
In
a
500
ml
erlenmeyer
flask
(Note
1),
equipped with
a
magnetic stirring device, was placed 100 ml of a 70%(v/v) solution
of conc. nitric acid-sulfuric acid.
The solution was stirred and
cooled in an ice bath for five minutes.
Hydroxyquinaldine (10 g,
over a five minute period.
62.9 mmol)
8-
was added in small portions
The addition of the 8-Hydroxyquinaldine
caused a brownish gas to evolve (Note 2).
to stir in the ice bath for 90 minutes.
with a hint of red.
To this solution,
The mixture was allowed
The mixture appeared black
The mixture was then poured into a 1000 ml
beaker which contained 600 ml of distilled water, and stirred with
a glass rod.
A bright yellow precipitate formed upon the addition
of the mixture to the water.
The precipitate was then either
vacuum filtered and collected, or it was allowed to stand overnight
and the supernate was carefully vacuum pipetted out with a pipet
attached
to
filtration.
the
aspirator,
and
followed
by
a
short
vacuum
The solid material was added to 150 ml of 95% ethanol.
The precipitate tended to cling to the filter paper, so the filter
paper was
rinsed with 50 ml
ethanolic mixture
of 95% ethanol
and added to
in a 500 ml erlenmeyer flask
(Note 3).
mixture was magnetically stirred for 15 minutes (Note 4).
the
The
This
solution was again filtered, and the solid on the funnel was washed
with ethyl ether (150 ml), with the aspirator off (Note 5).
,-
The
solid 5, 7-Dinitro-8-hydroxy-2-methylquinoline was left on the paper
11
-
to dry overnight in the hood.
The compound was a very bright
yellow.
A greenish tint shows impurity.
Note 1:
The use of a 500 ml flask, instead of a 250 ml flask, will
greatly lessen the chances of an intense evolution of gas, and an
overflowing of the mixture during the next procedure.
Note 2:
Be careful!
mixture may overflow.
Note 3:
If too much compound is added at once, the
Perform this procedure under g hood only.
The cleaner the filter paper, after rinsing, the higher
the yield.
Get the 'filter paper as clean as possible, sometimes
"dipping" the paper in ethanol helped to get the compound off of
the paper.
This compound is very "messy," latex gloves and a lab
coat are highly recommended.
Note 4:
Additional ethanol may have to be added while stirring to
wash the compound efficiently.
The amount of ethanol used here is
not crucial, since the solution will be filtered later.
Note 5:
The addition of the ether to the solid on the funnel
caused the paper to be raised up, and the ether seeped under the
paper, taking some of the product with it.
To avoid this, make
sure the filter paper is secure and tight on the funnel, with no
bubbles, before beginning the filtration.
If unsure of the status
of the filter paper, add the ether slowly, and do not allow it to
build up and flow around the edges.
Yield: 57.5%
-
(9.0 qrams of product)
Yields of 77% were reported by A.G.Richardson8
12
B. PREPARATION OF 5,7-DIACETAMIDO-2-METHYL-8-ACETOXYQUINOLlBE(13)
Into a 500 ml hydrogenation bottle, 5,7-Dinitro-8-hydroxy-2methylquinoline (6.03 g, 24.2 mmol) , water (100 ml), and conc. HCI
(13 ml) were added.
To this suspension, 5% Palladium on Charcoal
(2.00 g) was added as a catalyst.
40 psi overnight (Note 1).
filtered
to
remove
the
This mixture was hydrogenated at
The solution was then carefully vacuum
(Note
PdlC
transferred to a 500 ml beaker.
2)
and
the
filtrate
was
To the filtrate, sodium sulfite
(2.85 g), sodium acetate (3.58 g), and acetic anhydride (2.5 ml)
were added, using a magnetic stirrer.
formed.
After 30 minutes,
compounds were added again.
Generally, no precipitate
the same amount of the above three
The slow addition of acetic anhydride
(7.5 ml) caused a orange/white precipitate to form (Note 3).
The
solution was then vacuum filtered and the precipitate was washed
three times with water (100 ml).
This process washed the orange
color out of the precipitate, and left a white solid on the filter
paper.
volume.
above.
The filtrate was evaporated down to one-quarter of its
Any precipitate that formed was filtered and washed, as
The filtrate was treated with sodium acetate and acetic
anhydride aga in until no more precipitate was formed (Note 4).
All
washed precipitates were combined and allowed to dry overnight
under the hood, or on a vacuum pump.
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I
13
-
Note 1:
The hydrogenation should take place until no more of the
hydrogen gas is "used" by the reaction.
Therefore, do not stop the
hydrogenation until the rate of pressure decrease falls below 0.5
psi/hour.
Note 2:
water!
This takes approximately four hours.
Do Not rinse the Pd/C that
is on the filter paper with
This will dilute the solution,
sensitive.
which is concentration
The filtrate should take up, at most, 130 mI.
Note 3:
If after this point,
no precipitate has formed,
then
perform the following procedure:
Using a rotary evaporator and heat, remove the solvent until
a small amount of solution is obtained.
(sodium sulfite, sodium acetate,
quantities
Add the three compounds
and acetic anhydride)
in small
(approximately one-quarter of that called for in the
procedure),
and then wait
15
minutes.
Next
add
5 ml
acetic
anhydride and stir vigorously (use a magnetic stirrer, cover the
beaker with parafilm, and set the stirrer on maximum).
amounts
of
Add small
sodium sulfite and sodium acetate while vigorously
stirring for 10 minutes.
stop the stirring and allow the mixture
to stand, the precipitate should then form.
Problems like this can
be
procedure
avoided
by
following
the
prescribed
exactly
as
written.
Note 4:
The addition of the acetic anhydride is what generally
causes more precipitate to form.
Yield: 80% (6.05 grams of product [m.p. 255- C])
Yields of 88% were reported by A.G.Richardson8
14
-
C. PREPARATION OF 7-ACETAMIDO-2-KBTHYLQOINOLINE-5,8-DIONE(14)
Into a
1000 ml erlenmeyer flask,
240 ml of glacial acetic
acid, and 5,7-0iacetamido-2-methyl-8-acetoxyquinoline (6.3 g, 2.0
romol)
were added.
To the resulting suspension,
a
solution of
potassium dichromate (17.64 g) in 200 ml of water was added.
This
black soluticm was magnetically stirred at room temperature for 2024 hours.
The solution was then poured into 900 ml of water and
extracted with dichloromethane (5 x 200 ml).
The organic extracts
were washed with a solution of 5% sodium carbonate in a saturated
salt solution (3 x 300 ml).
The organic layer was dried overnight
with anhydrous magnesium sulfate (Note 1).
was
filtered
off,
orange/yellow solid.
and
the
solvent was
The magnesium sulfate
evaporated to
leave
a
The solid 7-Acetamino-2-methylquinoline-5,8-
dione was dried overnight on the vacuum pump.
Note 1:
The organic layer is more efficiently dried if the flask
is tightly stoppered, sealed with parafilm, and placed in a cold
room.
Yield: 55.65% (2.56 9 of product)
Yields of 53% were reported by A.G. Richardson8
15
-
D. PREPARATION OF 7-ACETAMlDO-2-FORMYLQUINOLlNE-S,8-DIONE(8)
In a dry 100 ml round bottomed flask, equipped with a magnetic
bar,
2.30 g
(10 mmol) of 7-acetamido-2-methylquinoline-5,8-dione
and 1.388 g (12.5 mmol) of selenium (IV) oxide (note 1) was added
to 35.0 ml of dried 1,4-dioxane (note 2) and 1.25 ml of water.
reaction mixture
period.
was heated slowly to
reflux
The mixture was then refluxed for 24 hours.
was carried out under an argon balloon.
reaction was tested by TLC
dioxane was
minutes.
over a
added
(note 13).
and was allowed to
The
two hour
This reaction
The completion of the
To the mixture 35 ml of
reflux
for
fifteen more
The solution was then hot filtered to remove the black
selenium metal.
The flask was rinsed with approximately 10 ml of
dichloromethane and poured over the solid on the filter paper.
fil trate was then rotary evaporated to dryness.
extracted with 2500 ml of dichloromethane.
The
The sol id was
The filtrate was then
rotary evaporated to obtain the yellow-tan aldehyde (7-acetamino-2formylquinoline-5,8-dione).
The product was dried on the vacuum
pump overnight and a TLC was performed (note 3).
product was yellow in color.
~
I
16
When dried, the
The purity and quality of the selenium (IV) oxide will
Note 1:
severely effect the yield.
pure~
Be sure the selenium (IV)
oxide is
If it is, it will appear as orange crystals.
Note 2:
Dioxane was refluxed over argon, adding KOH until no more
black resin appeared.
This took approximately 4-6 hours.
The
resin was filtered out and the dioxane was refluxed with sodium
spheres until they appeared shiny.
The dioxane was then distilled.
Dioxane was kept in an argon atmosphere in a bottle equipped with
a
septum,
and
removed with a
sterile
syringe,
using an argon
balloon to regulate the pressure.
Note 3: Use a solvent system of 100% EtOAc.
0.51-0.56 indicates completion.
Yield:
72~95%
An Rf value of approx.
There should be one spot.
(356 mg of product)
Yields of 80% were reported by A.G. Richardson8
17
E. SYNTHESIS OF 5-BYDROXYTRYPTOPHAN ISOAMYL ESTER (7)
Into a dry 250 ml round bottomed flask was added 60 ml of
dried and distilled isoamyl alcohol
(note 1).
Dry HCl gas was
bubbled through the isoamyl alcohol for 15 minutes.
The HCl tank
was opened so that it bubbled into the isoamyl alcohol through a
pipett in a side neck of a three necked round bottomed flask.
The
isoamyl alcohol was kept under an argon stream flowing throug a ITI
tube at the top of the condenser leading to a concentrated solution
of sodium hydroxide. To the saturated HCl solution was added 5(~
Hydroxy tryptophan
suspension.
-
150
2).
0
1
gram,
4.54
romol).
This
created
a
The reaction mixture was heated in an oil bath to 145
C. The colorless solution was refluxed for 24 hours (note
The solution was then allowed to cool at room temperature.
The mixture was evaporated to "dryness" under vacuum to give the
hydrochloride salt.
The oil material was dissolved in 40 ml EtOAc
and then was treated with 10 - 15 ml of conc. NH40H.
solution was recorded to be
stirred for 10 minutes.
~
9.
The pH of the
This mixture was magnetically
The organic layer was separated and the
aqueous layer was extracted with EtOAc (2 x 10 ml).
organic
extracts
solution.
were
washed
with
25
ml
of
a
The combined
saturated NaCl
The organic layer was separated and dried over anhydrous
MgS0 4 for 2 hours or overnight in the cold room.
The mixture was
filtered, and the solution was evaporated to dryness under vacuum.
Small portions of ethyl ether were added several times to the oil
,'-"
and then evaporated to help the removal of the EtOAc.
18
The TLC with
-
a solvent system of 9:1 EtOAc/MeOH showed one spot (Rf=0.51), but
the nmr showed traces of EtOAc.
To remove the EtOAc, the oil was
dried overnight on the vacuum pump at 70 C.
0
showed the product to be pure.
A subsequent nmr
The 5-Hydroxytryptophan isoamyl
ester was a dark brown, highly viscous oil, and was stored in the
cold room. 9
Yield: approx. 80\ (approx. 1 gram of product)
'H NMR (CDCI 3 , ppm) 8.03
(lH, s, NH), 7.265
(lH, s, C-8 H),
7.20 (lH, d, J=4.4 Hz, C-6 H), 7.013 (lH, s, C-4 H), 6.75 (lH, d,
J=0.91 Hz, C-7 H),
4.135 (2H, t, J=7 Hz, COzCHz-), 3.8 (lH, t, J=6
Hz, C-2 H), 2.95-3.25 (2H, m, HaHb style, C-3 H), 1.54-1.73 (lH, m,
-CH Z-) ' 1.47-1.53 (lH, rn, -CH'-), 0.896 (6H, d, J=3.1 Hz, C-(CH3 )z);
lR (Neat) vmax
3420 (O-H); 3359,3299 (N-Hz ); 3206 (N-H); 3054 (aromatic
2959,2929,2872(aliphatic
C -H) ;
1667,1626,1582 (aromatic
(CH 3 ) z
bendin~J);
intensity)
290
C=C);
1208 (Ar-O-);
(W,
6.3),
274
1468 (-CHz-
C-H) ;
bending);
1052 (-CHz-O-);
(1.5),
216
1729(C=O) ;
ElMS,
(7.4),
1386,1366 (C-
role
173
(relative
(1.6),
162
(1.3),158 (1.1), 146 (100), 117 (1.9).
Note 1:
The isoamyl alcohol (3-methyl-1-butanol) was dried over
anhydrous CuS04 and distilled.
addition of the CuS04 •
The alcohol turned brown upon the
The boiling range of the isoamyl alcohol is
130.5 - 131.5"C.
Note 2:
When the oil bath reached 145°C, the 5-Hydroxytryptophan
dissolved in the alcohol.
19
-.
F.
SYNTHESIS OF THE ISOAMYL ESTER OF 7-N-ACETYL-ll l -BYDROXY-3 1 DEMETHYLLAVENDAMYCIN(5)
Into a dry 150 ml round bottomed flask was added 75 ml of
dried and distilled acetonitrile (Note 1).
Hydroxy tryptophan
isoamyl
(1.,
ester
Acetamido-2-formylquinoline-5,8-dione
To this was added 5-
56
mg,
0.2
romol),
(~,
48.8 mg, 0.2 mmol).
7The
The lemon
reaction mixture was heated in an oil bath to 82°C.
yellow solution was refluxed for 19 hours.
and
The mixture was then
evaporated to dryness under vacuum to give a brown solid.
The
solid was then dissloved in a minimal amount of Chloroform and the
compound was separated by plate chromatography (Rf=0.23) to give
the red solid lavendamycin derivative.
Thin layer chromatography
(silica gel, CH2Cl 2 - MeOH, 100:1) showed the compound to be pure
(Rf=0.4) .
The
nmr
showed
traces
of
unreacted
ester,
the
lavendamycin derivative peaks are listed below (Note 2).
yield: 6.9%
'H NMR (CDCl 3 , ppm)
11.6(lH, s, NH), 9.16(lH, d, J=4.14 Hz,
C-4 H), 8.89(lH, s, C-6 H), 8.47 (lH, d, J=13.2 Hz, C-3 H), 8.4(lH,
s, AcNH),
7.97(lH, s, C-3
7.6(lH, s, C-12
1
1
H),
7.65(lH, d, J=0.91 Hz,
C-10 ' H),
H), 7.3(lH, d, J=7.4 HZ, C-9 1 H), 4.5(2H, t, J=6
Hz, C0 2CH2 ), 2.0-2.15 (2H, m, C-CH2-), 1.82(lH, q, J=6 Hz, -CH'-),
1.1(6H, d, J=6 Hz, C-(CH3 )z.
Note 1:
Acetonitrile was dried by distilling over calcium hydride.
Note 2:
The reaction was done twelve times and no pure compound
was made.
The starting materials MUST be pure,
and the water
should be removed effectively from the reaction flask.
20
REFERENCES:
1
Doyle, T.W., Balitz, D.M.,
"structure
Antitumor
Grulich, R.E., Nettleton,
Determination
Antibiotic
of
From
Tetrahedron Letters,
vol.
Lavendamycin
streptomyces
22,
no.
46,
D.E.,
A
New
Lavendulae,"
pp.
4595-4598,
1981.
2
Balitz,
D.M.,
Bush,
Lavendamycin,
A
J.A.,
New
Doyle,
T.W.,
Antibiotic
From
"Isolation
of
streptomyces
Lavendulae," The Journal of Antibiotics, vol. XXXV, no. 3,
March 1982, pp.259-265.
3
Zarrinmayeh, Hamideh,
"synthesis Of B-Carbolines And Their
Tetrahydro Derivatives," Master of Science Thesis at Ball
State University, Muncie, Indiana, August, 1986.
4
M. Behforouz, "Synthesis and study of ras" Specific Antitumor
Drugs," Grant Proposal, National Institutes of Health AREA, 1990.
5
Kende, A.S., Ebetino, F.H., "The Regiospecific Total Synthesis
Of
Lavendamycin Methyl
Ester,"
vol.25, no.9, pp.923-926, 1984.
21
Tetrahedron
Letters I
-
6
Gu,
Zhengxiang,
"Total
Synthesis
of
Lavendamycin
Methyl
Ester," Research report, Ball State University, 1989.
7
Behforouz,
Mohammad,
Zarr inrnayeh,
Hamideh,
Ogle,
Mark E.,
Riehle, Tammy J., Bell, Frank W., J. Heterocyclic Chern,
25,
8
1627 (1988).
Richardson, A.G., Synthesis of 7-acetamino-2-forrnylguinolineS,8-dione,
Ball
state
University
Chemistry
Dept.
Research report, 1991.
9
Cai, W., Synthesis of Tryptophan isoamyl methyl ester, Ball
State University Chemistry Dept. research notebook, 1991.
-
I
22
-
APPENDIX:
The following pages contain nmr I
ir,
and mass spectra
compound I, and an nmr spectrum of compound
23
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2.3131
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Tom Eads Tryp-Ester EI 4-10-92.scan TIC
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Tom Eads Tryp-Ester EI 4-10-92.scan scan 16 - 126
1
12651
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Tom Eads Tryp-Ester EI 4-10-92.scan scan 12 - 20
1 .0
6387
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til
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4
5
Mass
Tom Eads Tryp-Ester EI 4-10-92.scan scan 59 - 65
.....o
14 .0
28007
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1
5
Mass
Tom Eads Tryp-Ester EI 4-10-92.scan scan 68 -71
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44732
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Tom Eads Tryp-Ester EI 4-10-92.scan scan 100 - 102
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58282
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Mass
Tom Eads
Tryp-Ester
EI
4-10-92.scan scan 100 - 102
Fri Apr 10 11:11:11 1992
-
Mass
Intensity
I%
101.0
102.0
103.0
104.0
105.0
106.0
107.0
108.0
109.0
110.0
111. 0
112.0
114.0
115.0
116.0
117.0
120.0
121.0
122.0
123.0
124.0
125.0
126.0
127.0
128.0
129.0
130.0
131.0
133.0
134.0
136.0
140.0
141.0
142.0
143.0
144.0
145.0
146.0
150.0
151.0
153.0
154.0
155.0
156.0
158.0
159.0
160.0
161. 0
162.0
163.0
164.0
165.0
167.0
168.0
169.0
170.0
171.0
238
238
358
206
136
55
66
117
120
22
35
104
14
70
362
1123
61
41
20
61
6
70
173
404
272
101
359
136
324
79
17
23
48
50
61
525
336
58282
9
6
21
12
125
168
668
481
256
21
780
164
236
152
146
9
15
31
124
0.4
0.4
0.6
0.4
0.2
0.1
0.1
0.2
0.2
0.0
0.1
0.2
0.0
0.1
0.6
1.9
0.1
0.1
0.0
0.1
0.0
0.1
0.3
0.7
0.5
0.2
0.6
0.2
0.6
0.1
0.0
0.0
0.1
0.1
0.1
0.9
0.6
100.0
0.0
0.0
0.0
0.0
0.2
0.3
1.1
0.8
0.4
0.0
1.3
0.3
0.4
0.3
0.3
0.0
0.0
0.1
0.2
~
-
172 .0
173.0
174.0
175.0
178.0
182.0
185.0
186.0
188.0
190.0
191. 0
194.0
195.0
198.0
199.0
202.0
203.0
204.0
206.0
207.0
208.0
213 .0
214.0
215.0
216.0
219.0
222.0
224.0
228.0
229.0
231.0
239.0
242.0
244.0
245.0
246.0
252.0
254.0
258.0
259.0
261. 0
266.0
269.0
270.0
271. 0
272 .0
274.0
275.0
276.0
281. 0
284.0
285.0
286.0
288.0
289.0
290.0
292.0
301. 0
302.0
252
916
334
453
15
6
36
63
48
18
53
16
11
12
21
34
68
275
10
278
19
12
173
43
4314
41
8
10
8
23
62
17
5
188
212
42
31
10
126
24
11
4
3
4
6
73
858
61
13
31
3
15
9
95
102
3696
26
97
27
0.4
1.6
0.6
0.8
0.0
0.0
0.1
0.1
0.1
0.0
0.1
0.0
0.0
0.0
0.0
0.1
0.1
0.5
0.0
0.5
0.0
0.0
0.3
0.1
7.4
0.1
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.3
0.4
0.1
0.1
0.0
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.1
1.5
0.1
0.0
0.1
0.0
0.0
0.0
0.2
0.2
6.3
0.0
0.2
0.0
305.0
5
315.0
330.0
343.0
344.0
357.0
0.0
22
16
9
30
4
0.0
0.0
0.0
0.1
0.0
--
358.0
360.0
361.0
362.0
434.0
436.0
13
105
141
47
4
3
0.0
0.2
0.2
0.1
0.0
0.0
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